Within the biotechnology industry, the purification of proteins on a commercial scale is an important challenge to the development of recombinant proteins for therapeutic and diagnostic purposes. Problems related to yield, purity, and throughput challenge the manufacturing sector. With the advent of recombinant protein technology, a protein of interest can be produced using cultured eukaryotic host cell lines engineered to express a gene encoding the protein. What can result from a host cell culturing process, however, is a mixture of the desired protein along with impurities that are either derived from the protein itself, such as protein variants, or from the host cell, such as host cell proteins, DNA, and cellular debris. The use of the desired recombinant protein for pharmaceutical applications may be contingent on being able to reliably recover adequate levels of the protein from these impurities. Recombinant technology can also produce proteins that are not found in nature, for example, novel mutant proteins, fusion proteins, or proteins with heterologous signal sequences that direct the secretion of the protein to the medium. Recombinant proteins can be expressed in many eukaryotic cell types, including Chinese Hamster Ovarian cells (CHO), baby hamster kidney (BHK), NS0 myeloma cells, and Pichia pastoris yeast cells.
Typically, to produce a recombinant protein, a recombinant DNA vector is created that contains a gene that codes for the protein to be expressed with appropriate sequences to direct the transcription and translation of the gene in the desired cell type. The vector can also contain sequences such as selectable or counterselectable markers, for example, drug resistance genes, and/or sequences designed to promote the stable retention of the protein expression sequences. For mammalian cells, plasmid and viral vectors, for example, retroviral vectors, can be used.
Following the creation of the vector, the vector is then introduced into the cells. The vector can be transfected as naked DNA using standard methods, for example, lipofection, calcium phosphate, DEAE-dextran, electroporation, or biolistics (gene gun). Viral vectors can be introduced by infection with viral particles. The cells are then screened or selected for those that contain the vector.
Cells that contain the vector and express the recombinant protein can be grown in a liquid medium or on a solid support, and the protein isolated from the cell culture. Mammalian cell density ranges between 106 cells/mL to 2×107 cells per mL or more. Most proteins are secreted. Secreted protein concentrations can range between 4 mg/L to 10 g/L. However, if the protein is produced intracellularly, the cells are broken to release the protein, whereas if the protein is secreted, it can be isolated from the growth medium or the support following removal of the cells and cell debris. The isolated protein can then be purified.
Conventional biopharmaceutical protein purification methods used to remove cells and cellular debris include centrifugation, microfiltration, and depth filters. Filter aids, such as diatomaceous earth, can be used to enhance performance of these steps, but they are not always effective and sometimes significantly bind the product of interest. Their use may also require the addition of a solid or a homogeneous suspension that can be challenging as part of large-sale biopharmaceutical operations.
Polymeric flocculants can be used to aid in the clarification of mammalian cell culture process streams, but they can have limitations. For example, protamine sulfate preparations typically used as processing aids are limited in application due to concerns about inactivation of the protein of interest or product loss due to precipitation (Scopes, 1987). High quality reagent, such as that sold for medical use, can be expensive. In certain instances, removal to very low levels may require validation to ensure there are no unexpected effects in patients. For example, chitosan is not a well-defined reagent and there are concerns about its consistent performance in routine use in clarification applications. Multiple charged polymers, such as DEAE dextran, acrylamide-based polymers often used in waste-water treatment (NALCO Water Handbook, Chapter 8) and polyethylene amine (PEI) have been considered for use in clarification applications. With respect to the latter two types of polymers, the acrylamide reagents have the potential for contamination with toxic reagents and polyethylene amine, while a highly effective clarification reagent, is often contaminated with varying amounts of ethylenimine monomer, a suspected cancer agent (Scawen et al). Moreover, many of these polymers, including PEI, tend to bind almost irreversibly to many chromatography resins, thereby limiting downstream processing options. The regulatory and raw material reuse concerns associated with these polymers have limited their application primarily to academic studies.
Non-polymer based flocculants, such as alum and iron salts, have been utilized in the wastewater treatment industry (NALCO Water Handbook). These substances may appear to be non-useful in processing protein products, because they may bind to the protein product or may catalyze chemical reactions resulting in modifications of the protein that could affect safety or efficacy.